Engineering ML-IAP to produce an extraordinarily potent caspase 9 inhibitor: implications for Smac-dependent anti-apoptotic activity of ML-IAP

Biochem J. 2005 Jan 1;385(Pt 1):11-20. doi: 10.1042/BJ20041108.


ML-IAP (melanoma inhibitor of apoptosis) is a potent anti-apoptotic protein that is strongly up-regulated in melanoma and confers protection against a variety of pro-apoptotic stimuli. The mechanism by which ML-IAP regulates apoptosis is unclear, although weak inhibition of caspases 3 and 9 has been reported. Here, the binding to and inhibition of caspase 9 by the single BIR (baculovirus IAP repeat) domain of ML-IAP has been investigated and found to be significantly less potent than the ubiquitously expressed XIAP (X-linked IAP). Engineering of the ML-IAP-BIR domain, based on comparisons with the third BIR domain of XIAP, resulted in a chimeric BIR domain that binds to and inhibits caspase 9 significantly better than either ML-IAP-BIR or XIAP-BIR3. Mutational analysis of the ML-IAP-BIR domain demonstrated that similar enhancements in caspase 9 affinity can be achieved with only three amino acid substitutions. However, none of these modifications affected binding of the ML-IAP-BIR domain to the IAP antagonist Smac (second mitochondrial activator of caspases). ML-IAP-BIR was found to bind mature Smac with low nanomolar affinity, similar to that of XIAP-BIR2-BIR3. Correspondingly, increased expression of ML-IAP results in formation of a ML-IAP-Smac complex and disruption of the endogenous interaction between XIAP and mature Smac. These results suggest that ML-IAP might regulate apoptosis by sequestering Smac and preventing it from antagonizing XIAP-mediated inhibition of caspases, rather than by direct inhibition of caspases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Amino Acid Sequence
  • Apoptosis Regulatory Proteins
  • Apoptosis*
  • Binding Sites
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Caspase 9
  • Caspase Inhibitors*
  • Caspases / metabolism
  • Cell Line
  • Cell Line, Tumor
  • Crystallography, X-Ray
  • Cysteine Proteinase Inhibitors / chemistry*
  • Cysteine Proteinase Inhibitors / genetics
  • Cysteine Proteinase Inhibitors / metabolism*
  • Electron Spin Resonance Spectroscopy
  • Humans
  • Inhibitor of Apoptosis Proteins
  • Intracellular Signaling Peptides and Proteins
  • Kinetics
  • Mitochondrial Proteins / chemistry
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Neoplasm Proteins / chemistry
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism*
  • Protein Binding
  • Protein Engineering*
  • Protein Structure, Tertiary
  • Proteins / chemistry
  • Proteins / genetics
  • Proteins / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • X-Linked Inhibitor of Apoptosis Protein


  • Adaptor Proteins, Signal Transducing
  • Apoptosis Regulatory Proteins
  • BIRC7 protein, human
  • Carrier Proteins
  • Caspase Inhibitors
  • Cysteine Proteinase Inhibitors
  • DIABLO protein, human
  • Inhibitor of Apoptosis Proteins
  • Intracellular Signaling Peptides and Proteins
  • Mitochondrial Proteins
  • Neoplasm Proteins
  • Proteins
  • Recombinant Fusion Proteins
  • X-Linked Inhibitor of Apoptosis Protein
  • XIAP protein, human
  • CASP9 protein, human
  • Caspase 9
  • Caspases